21 Adventitious root (AR) formation at the base of stem cuttings determines the efficiency 22 of clonal propagation for woody plants. Many endogenous and environmental factors 23 influence AR formation. However, our knowledge about the regulation of AR 24 development by mitochondrial metabolism in plants is very limited. Here we identified 25 Populus-specific miR476a as a novel regulator of wound-induced adventitious rooting 26 via orchestrating mitochondrial homeostasis in poplar. MiR476a exhibited inducible 27 expression during AR formation and directly targets several Restorer of Fertility like 28 (RFL) genes encoding mitochondrion-localized pentatricopeptide repeat proteins. 29 Genetic modification of miR476-RFL expression revealed the miR476/RFL-mediated 30 dynamic regulation of mitochondrial homeostasis on AR formation in transgenic 31 poplar. Furthermore, mitochondrial perturbation via exogenous chemical inhibitor 32 validated that the miR476a/RFL-directed AR formation depended on mitochondrial 33 regulation though modulating the auxin pathway. Our results established a 34 miRNA-directed mitochondrion-auxin signaling cascade required for AR development, 35 providing novel insights into the understanding of mitochondrial regulation on plant 36 developmental plasticity. 37 38